CN101351563A - Method to predict or monitor the response of a patient to an ErbB receptor drug - Google Patents

Abstract

The invention provides a method for detecting ErbB receptor mutations comprising the steps of providing a bio-fluid sample from a patient; extracting DNA from said sample; and screening said DNA for the presence of one or more mutations that alter tyrosine kinase activity in the receptor.

Description

Be used to predict or monitor the method for patient for the response of ErbB receptor agents

Technical field

The present invention relates to a kind ofly be used for prediction or monitoring patient for the ErbB receptor agents, for example is the Gefitinib (gefitinib) of target spot with EGF-R ELISA (EGFR), the method for response.This method provides sensitivity and the special screening that is used for genomic dna sudden change, and this sudden change appears at biofluid for example in the serum with lower concentration.This method is suitable for detecting the relevant sudden change of response that those known increase ErbB tyrosine kinase receptors are active and appear and the ErbB receptor agents is treated.

Background technology

The ErbB acceptor is protein tyrosine kinase (TKs), belongs to the TK superfamily, the signal conduction of this superfamily member regulation and control control cell growth and existence by way of.The ErbB receptor family is made of four kinds of closely-related hypotypes: ErbB1 (EGF-R ELISA [EGFR]), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (Cell.2000; 103:211-225).

For example, from the signal of EGFR be by somatomedin for example Urogastron (EGF) in conjunction with what cause, cause the EGFR molecule dimerization or with other closely-related acceptor different dimerization of HER2/neu for example.The recruitment that autophosphorylation that this receptor is undertaken by their tyrosine kinase domain and transphosphorylation cause the downstream effect device and the activation (Exp.Cell.Res.2003 of hyperplasia and cell survival signal; 284:31-53).When ErbB acceptor TKs is crossed when being expressed or being activated by sudden change, it can cause the development (Exp.Cell.Res.2003 of mammary cancer, nonsmall-cell lung cancer (NSCLC), colorectal carcinoma, head and neck cancer and many other noumenal tumours; 284:122-130).EGFR is crossed in the nonsmall-cell lung cancer of 40-80% and many other epithelial cancers expresses (N.Engl.J.Med.2004; 350 (21): 2129-2139).With the product of such gene is that the anticancer therapy of target spot has been devised the activity that is used to suppress them.For example, the medicine Gefitinib is for example powerful inhibitor of ErbB1 of EGFR family tyrosine kinase, and is approved in the treatment of inoperable or recidivity NSCLC in Japan on July 5th, 2002.

Patient is different for replying of any prescription drugs at it, comprises its effect how good (validity of medicine) and to its untoward reaction (side effect) two aspects.For the situation of Gefitinib, patient demonstrates different responses for treatment with tyrosine kinase inhibitors, is included in the group that about 10% patient demonstrates fast and often is tangible clinical response (N.Engl.J.Med.2004; 350 (21): 2129-2139).Therefore need before treatment, will discern the patient that medicine responds, and need the treatment back that those are discerned the patient that medicine responds, thereby make medicine more effectively by target to those.

Recent findings in the patient's who suffers from nonsmall-cell lung cancer group, is carried specific sudden change in the patient EGFR gene, and it appears to be associated with clinical response for the tyrosine kinase inhibitor Gefitinib, and (Science 2004; 304:1497-1500).These sudden changes have caused the growth of growth factor signal, make it for the inhibitor sensitivity.It is believed that these sudden changes in the screening lung cancer can identify the patient (J.Clin.Oncol.23 that those will have response for Gefitinib; 2493-2501).Yet up to now, the unique method of measuring these sudden changes reliably is by taking out patient's tumor biopsy sample the solid tissue sample to be analyzed.This is the program of a difficulty, is very offending for patient, and is impracticable when tumour should not be performed the operation sometimes.

The problem of another screening patient sudden change is to detect the difficulty of mutator gene in too much wild type gene.This is a problem well known in the art, and the identification of lower concentration mutant DNA for the early detection of tumour or suitably treat for early stage patient the identification of time-histories may very important prerequisite under, this particularly important (Clin Cancer Res.2004 that seems; 10 (7): 2379-85).Therefore, need less invasive and reliable more method to monitor and predict the response of patient for the ErbB receptor agents, for example use before them in treatment, this treatment may be very effective, but only effective to the sub-fraction patient.

Summary of the invention

We have found that a kind of method that can detect ErbB receptor mutation in the biologicfluid sample that picks up from patient reliably, this method can be used for predicting response or the survival advantage (survival benefit) of patient to the ErbB receptor agents.Particularly, the existence of the sudden change of the tyrosine kinase activity of those changes ErbB acceptor points out that patient may have positive response to medicine, only exists wild-type allele to point out that patient may not can have response to the ErbB receptor agents simultaneously.

According to a first aspect of the present invention, a kind of method that is used to detect the ErbB sudden change is provided, comprise step :-

(a) provide biologicfluid sample from patient

(b) from described sample, extract DNA; With

(c) screen described DNA at the situation that exists of one or more sudden changes in this receptor (one or more mutations).

Preferably, the aforesaid method that is used for detecting the ErbB sudden change is included in the one or more detections that can change the sudden change of tyrosine kinase activity in the described acceptor of ErbB acceptor.

Most preferably, the ErbB acceptor of aforesaid method description is EGFR.

The inventor has been found that the measurement of the sudden change in patient's biologicfluid sample can be used to predict and monitor ErbB receptor agents effect in vivo.

One preferred aspect, the invention provides a kind of method of patient that be used to predict for the response of ErbB receptor agents, comprise step :-

(a) provide biologicfluid sample from patient

(b) from described sample, extract DNA

(c) screen described DNA at the situation that exists that changes one or more sudden changes of tyrosine kinase activity in this receptor

In another embodiment of the invention, a kind of method of patient for the response of ErbB receptor agents that be used to monitor is provided, comprise step:

(a) provide biologicfluid sample from patient

(b) from described sample, extract DNA

(c) screen described DNA at the situation that exists of one or more sudden changes that can change tyrosine kinase activity in this receptor.

As will be, allow the replying of patient that is given medicine made evaluation for the monitoring of the response of ErbB receptor agents by known to these those skilled in the art; Therefore, the patient after it can be used to treat.Yet the prediction of response is to carry out in the patient who does not contact the ErbB receptor agents, and carries out before the treatment.

In another embodiment, this method comprises aforesaid step, and wherein cancer patient has been predicted patient's survival advantage for the prediction of the response of ErbB receptor agents.

Preferably, the Forecasting Methodology of aforesaid response for the ErbB medicine further comprises step:

(d) conclude: be detected the patient that carries mutant and wild-type allele to the response of ErbB receptor agents with positive, can not make the male response and be detected the patient who only carries wild-type allele to this medicine.

In another embodiment, aforesaid screening method comprises the use polymerase chain reaction, and this polymerase chain reaction uses and detects the allele-specific primers that single base mutation, little in-frame deletion or base are replaced.

Preferably, screening method relates to and uses real-time polymerase chain reaction (real time-PCR), and this real-time polymerase chain reaction uses and detects the allele-specific primers that single base mutation, little in-frame deletion or base are replaced.

In another embodiment, for the Forecasting Methodology of the response of ErbB medicine as mentioned above, wherein first primer is to being used to detect wild-type allele, and second primer is to being used to detect mutation allele; And wherein every centering primer comprises :-

(a) has the primer that specific sudden change is had allele specific 3 ' terminal nucleotide; With

(b) the possible additional mispairing of this primer 3 ' end.

Preferably, every centering primer further comprises as mentioned above :-

(a) contain primer sequence and to the unit molecule or nucleic acid duplex (duplex) probe of other special sequences of target sequence;

(b) the fluorescence report dyestuff that be attached to probe 5 ' end close within described unit molecule or nucleic acid duplex with quenching medium (quencher) molecule;

(c) at the one or more non-coding nucleotide residue of described probe one end;

(d) wherein, described report dyestuff separates during the amplification of target sequence with the quenching medium molecule.

Advantageously, this probe is

Probe.

Preferably, method is used the technology that can detect the mutant nucleotide sequence that exists with wild-type sequence 10% level as described in the present invention.More preferably, this technology can detect the mutant nucleotide sequence that accounts for wild-type sequence 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1% or 0.01% level.

The benefit of aforesaid fluorescent probe system do not need to be independent probe to combine with the target of amplification, make detect rapider more effective again than other system.The present invention's proof is used in the ARMS amplification system

Primer has improved the susceptibility (seeing embodiment 4) of the method that is used to detect the EGFR sudden change.

Preferably, the biofluid of aforesaid method description is any in blood, serum, blood plasma, sweat or the saliva.Advantageously, biofluid is a serum.

Be used for retrospective research, the existence that studies have shown that sudden change such in the tumor sample of the excision that begin treatment is obtained later on that is conceived to EGFR sudden change and NSCLC progress dependency before the great majority.Yet the difficulty of taking a sample from inoperable NSCLC tumour of early stage patient has hindered the effort that had the expection research of selecting patient's potentiality before the treatment beginning.

Yet, the invention provides a kind of method that is used to detect from the sudden change EGFR of cancer patient sample, this sample is not a tumor sample.The sampling of biofluid is littler than the invasive of the method for former analysis cancer patient EGFR sudden change.Compare with collecting tumor sample, for example serum sample can easily be gathered, and test can be repeated.Known cancer cell released dna enters circulation in addition, is enriched in serum and the blood plasma, thereby allows to detect sudden change and small change (microsatellite alterations) (Cancer Res.1999 in the serum DNA of cancer patient; 59 (1): 67-70).

In another embodiment of the invention, the ErbB receptor agents is the ErbB receptor tyrosine kinase inhibitors.Preferably, the ErbB receptor agents is the EGFR tyrosine kinase inhibitor.In a preferred method, the EGFR tyrosine kinase inhibitor is selected from by Gefitinib, erlotinib (erlotinib) (Te Luokai (Tarceva), OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinibditosylate (lapatinib) (GW2016, GW-572016, GSK572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626, ZD6474 (Zactima ^TM) or WO2004/006846 or WO2003/082290 in the group that constitutes of disclosed any compound.

In another embodiment of the invention, the ErbB receptor agents is the EGFR inhibitor.Preferably, the EGFR inhibitor be selected from by Cetuximab (cetuximab) (Erbitux (Erbitux), C225), Herceptin (matuzumab) (EMD-72000), handkerchief Buddhist nun monoclonal antibody (panitumumab) (ABX-EGF/rHuMAb-EGFR), the anti-egfr antibodies of the group that constitutes of MR1-1, IMC-11F8 or EGFRL11.

The method of the claim before preferably, any comprises as single therapy or with other drug unites the ErbB receptor agents of use.

In the most preferred embodiment, EFGR tyrosine kinase inhibitor medicine is selected from by Gefitinib, erlotinib (Te Luokai, OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinibditosylate (GW2016, GW-572016, GSK572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626, ZD6474 (Zactima ^TM) or the group that constitutes of disclosed any compound of WO2004/006846 or WO2003/082290.

Found that the sudden change among the present invention takes place as insertion, disappearance or the replacement of nucleic acid.Sudden change preferably betides the tyrosine kinase domain of ErbB acceptor.Preferably, sudden change betides the tyrosine kinase domain of EGFR.Preferably, sudden change is selected from the group of the EGFR sudden change that is listed in the table 5.Advantageously, sudden change bunch collection is near the ATP-binding site of EGFR exons 18,19,20 or 21.Preferably, sudden change is selected from the group of the EGFR sudden change that is listed in the table 5.In the most preferred embodiment, sudden change is E746 A750del in the EGFR exons 19 and the L858R in the EGFR exon 21.

About 30 sudden changes among the EGFR exons 1 8-21 are detected in the lung tumor sample.During the EGFR that the NSCLC-that detects in tumor sample is relevant suddenlyd change, the point mutation (L858R) that the in-frame deletion (E746_A750del) of the 15-bp Nucleotide in the exons 19 and the leucine of the codon in exon 21 858 are replaced by arginine accounted for about 90% (Cancer Res.2004 of these sudden changes; 64:8919-8923, Proc.Natl.Acad.Sci USA2004; 101:13306-13311).

Advantageously, the cancer that patient suffered from is selected from by non-noumenal tumour for example leukemia, multiple myeloma or lymphoma, and the noumenal tumour group that constitutes of bile duct, bone, bladder, brain/CNS, glioblastoma, breast, colorectum, uterine cervix, uterine endometrium, stomach, head and neck, liver, lung, muscle, neurone, oesophagus, ovary, pancreas, pleura/peritonaeum, prostate gland, kidney, skin, testis, Tiroidina, uterus and external genital tumor for example.

In another embodiment of the invention, aforesaid method further comprises step:

(e) screen described DNA at the situation that exists of the one or more sudden changes in the element of ErbB acceptor downstream signal pathway.

A second aspect of the present invention comprises composition, and it comprises first primer of being used to detect ErbB acceptor wild-type allele to be used to detect allelic second primer of ErbB receptor mutation right, and wherein, a primer of every centering further comprises:

(a) have the primer of 3 ' terminal nucleotide, it is allele specific oligonucleotide for specific sudden change;

(b) the possible additional mispairing of this primer 3 ' end;

(c) comprise primer sequence and the unit molecule or the nucleic acid duplex probe that are specific to the other sequence of target sequence;

(d) the fluorescence report dyestuff that be attached to 5 ' terminal probe close within described unit molecule or nucleic acid duplex with the quenching medium molecule;

(e) at the one or more non-coding nucleotide residue of described probe one end;

(f) wherein, described report radical dye separates during the target sequence amplification with the quenching medium molecule.

The 3rd aspect of the present invention comprises the purposes in the analysis that the primer that is specific to the ErbB acceptor carries out in biofluid, be used to predict the response of patient for the ErbB medicine.

Preferably, aforesaid purposes comprises the manufacturing that is used for test organisms fluidic composition, and it is used to predict the response of patient for the ErbB medicine.

Advantageously, the purposes of foregoing description further comprises step:

(a) from described sample, extract DNA

(b) screen described DNA at the situation that exists that changes one or more sudden changes of tyrosine kinase activity in this receptor.

Description of drawings

Fig. 1 uses EGFR Scorpion test kit to detect the susceptibility of E746_A750del and L858R sudden change.(a) use the standard DNA with E746_A750del of different amounts: 10,000pg (10 ⁴), 1,000pg (10 ³), 100pg (10 ²), 10pg (10 ¹) and 1pg (10 ⁰).In same test, wild-type standard DNA (wild) and distilled water (D.W.) are used as negative control.(b) concentration is 1pg to 10, the standard DNA with E746_A750del and 10 of 000pg, and the wild-type standard DNA of 000pg was at 1: 1 (10 ⁰), 1: 10 (10 ^-1), 1: 100 (10 ^-2), 1: 1,000 (10 ^-3) and 1: 10,000 (10 ^-4) ratio under mix.(c) virgin curve and second curve of deriving represents 10, the standard DNA with E746_A750del of the amount of 000pg.Second derives represents the rate of change of growth curve slope.Threshold circulation is defined in second cycle number (vertical line among Fig. 1 c) of peak-peak of deriving curve.(d) typical curve is mapped to the log value of standard DNA amount from the Ct of every curve (shown in Figure 1A and 1B).

Fig. 2 picks up from the detection of the E746_A750del in the genomic dna of lung cancer cell line.(a) have PC-9 and the wild-type A431 of E746_A750del.(b) has the 11_18 of L858R and A431

Fig. 3 (A) and totally survives (overall survival) (B) about the existence that gets nowhere (Progression free survival) of the EGFR mutation status of nonsmall-cell lung cancer.(*) Log-rank test.

Embodiment

Unless otherwise defined, otherwise the implication of all technology used herein and scientific terminology all with the implication identical (for example in cell cultures, molecular genetics, nucleic acid chemistry, hybridization technique and biological chemistry) of those of ordinary skills' common sense.Standard technique is used for molecule, gene and biochemical method.Prevailingly referring to people such as Sambrook, Molecular Cloning:ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and people such as Ausubel, Short Protocols inMolecular Biology (1999) 4th Ed, John Wiley ﹠amp; Sons, Inc.; And people such as Guthrie, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Vol.194, Academic Press, Inc., (1991), (Innis waits people 1990.Academic Press to PCR Protocols:A Guide to Methods and Applications, San Diego, Calif.), people such as McPherson, PCR Volume 1N.Y.), and GeneTransfer and Expression Protocols, pp.109-128, ed.E.J.Murray, TheHumana Press Inc., Clifton, N.J.).These documents are included in this by reference.Oxford?University?Press，(1991)，Culture?of?Animal?Cells：A?Manual?ofBasic?Technique，2nd?Ed.(R.I.Freshney.1987.Liss，Inc.New?York，N.Y.)，and?Gene?Transfer?and?Expression?Protocols，pp.109-128，ed.E.J.Murray，The?Humana?Press?Inc.，Clifton，N.J.)。These documents are included in this by reference.

Biological marker

The various biological sign that is called as biological marker is identified and studies in the process that biological chemistry and molecular biology is applied to medical science and toxicology state.Biological marker can be found in tissue and the biofluid, wherein blood be in the biological marker research modal biofluid (Proteomics 2000; 1:1-13, Physiol.2005; 563:23-60).

Biological marker can be described to " indicator of the pharmacology response of interfering as normal biological processes, pathogenic course or to treatment and by the feature of measuring objectively and assessing ".Biological marker be anyly be identified and measure, with the specific state or the indicator of disease-related connection, wherein the existence of this biological marker or level and this state or disease in a certain respect between relevant (comprise existence, level or change level, type, stage, the susceptibility of described state or disease or for the response of the medicine that is used for the treatment of described state or disease).This association can be qualitatively, quantitative or both qualitative and quantitative.Typically, biological marker is compound, compound fragment or compound group.These compounds can be any compounds of finding or produce in the organism, comprise protein (and peptide), nucleic acid and other compound.

Biological marker can have Deuteronomic ability, so can be used to predict or detect existence, level, type or the stage (existence or the level that comprise specific microorganism or toxin) of specific state or disease, to specific state or the susceptibility of disease (susceptibility that comprises heredity) or to the response (comprising pharmacological agent) of specific treatment.It is believed that biological marker will play the effect that becomes more and more important to the discovery and the development of medicine by the efficient of improving research and development project in future.Biological marker can be used as the watch-dog of diagnostic reagent, disease progression, the watch-dog of treatment and the predictor of clinical effectiveness.For example, different biological marker research project is attempting discerning the sign of specific cancer and specific cardiovascular and Immunological diseases.

The term of Shi Yonging " ErbB receptor agents " comprises erbB receptor tyrosine kinase family is comprised the medicine that EGFR, ErbB2 (HER), ErbB3 and ErbB4 work herein.In one embodiment, this ErbB receptor agents is the ErbB receptor tyrosine kinase inhibitors.In another embodiment, this ErbB receptor agents is the EGFR tyrosine kinase inhibitor.The example of EGF receptor tyrosine kinase inhibitors includes, but are not limited to Gefitinib, erlotinib (OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinibditosylate (GW2016, GW-572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626 or disclosed any compound in WO2004/006846, WO2003/082831 or WO2003/082290.Particularly, disclosed Gefitinib (has another name called Iressa in International Patent Application WO 96/33980 ^TM(Iressa ^TM), code name is that ZD1839 and Chemical Abstracts registration number are 184475-35-2) be EGF-R ELISA (EGFR) the family powerful inhibitor of ErbB1 for example of Tyrosylprotein kinase.

In another embodiment, this ErbB receptor agents is for example a kind of among cetuximab (C225), matuzumab (EMD-72000), handkerchief Buddhist nun monoclonal antibody (ABX-EGF/rHuMAb-EGFr), MR1-1, IMC-11F8 or the EGFRL11 of anti-egfr antibodies.The ErbB receptor agents of herein mentioning can be used as single therapy or be used in combination with other similar or different classes of medicine.In a specific embodiments, this EGF receptor tyrosine kinase inhibitors is a Gefitinib.

" existence " comprise patient's " overall existence " and " existence gets nowhere "." overall existence " (OS) is defined as from beginning to give the time of Gefitinib to the death that is caused by any reason." existence (PFS) gets nowhere " is defined as from beginning to give the time of Gefitinib to the death that PD occurs for the first time or caused by any reason.

" response " relates to patient is divided into two main groups by according to " the response judgement criteria of solid tumor (Response EvaluationCriteria in Solid Tumours) " (RECIST) and the measurement of carrying out is defined: those demonstrate patients of partial response or stable disease and those demonstrate the patient of the sign of PD.

" amplification " reaction is those nucleic acid reactions that cause the specific amplification of target nucleic acid rather than non--target nucleic acid.Polymerase chain reaction (PCR) is well-known amplified reaction.

" cancer " of Shi Yonging is meant by cell to the conversion of tumor phenotypes (neoplastic phenotype) and the tumor growth that causes herein.Such cell transformation often relates to transgenation; In the context of the present invention, said, transform and to relate to the transgenation that the change by one or more Erb genes causes.

Term " probe " is meant the sequence specific oligonucleotide of strand, its sequence and allelic target sequence complementation fully to be detected.

Term " primer " is meant single strand dna oligonucleotide sequence or special primer, its can as with the synthetic starting point of nucleic acid chains complementary primer extension product to be duplicated.The length of primer and sequence must make them can start the synthetic of extension products.

The application has described the ErbB nucleic acid mutation.The nucleic acid that is used for any ErbB tyrosine kinase receptor of presentation code family member herein as the term " ErbB acceptor mutant " that uses.Term " ErbB acceptor " therefore comprises whole known human ErbB receptor homolog things and varient, and other nucleic acid molecule, and this nucleic acid molecule demonstrates and enough homologys that will be identified as the ErbB receptor family member of ErbB receptor homolog thing.Preferably, EGFR is identified as the nucleic acid with EGFR sequence shown in the SEQ ID NO.1.

Term " nucleic acid " comprise those under stringent hybridization condition can with the above-mentioned natural polynucleotide that have the hybridization of nucleic acid or its complement that are identified." stringent hybridization condition " is meant 42 ℃ of cultivations overnight in the solution of the salmon sperm dna that comprises 50% methane amide, 5x SSC (750mM NaCl, 75mM Trisodium Citrate), 50mM sodium phosphate (pH7.6), 5x Denhardt ' s solution, 10% T 500 and 20pg/ml sex change and shear, and then in 0.1x SSC at about 65 ℃ of washing filters.

Be used to detect the method for nucleic acid

In the context of the present invention, the detection of the mutant nucleic acid of coding ErbB acceptor can be used to predict the response for pharmacological agent.Because the sudden change in the ErbB acceptor gene usually occurs in dna level, method of the present invention can be based on the sudden change in the genomic dna and transcription product and their detection of protein.For the sudden change that guarantees to be detected is expressed among the experimenter really, confirm that by the analysis of transcription product and/or polypeptide the sudden change in the genomic dna may be a desirable.

Sudden change in the genomic nucleic acids advantageously detects from the nucleic acid fragment of amplification by the technology that changes (mobility shift) based on mobility.For example, Chen et al., Anal Biochem1996 Jul 15; 239 (1): 61-9 has described by competitive Mobility Shift Assay and has detected single base mutation.In addition, can also obtain the al. based on Marcelino et from the market, BioTechniques 26 (6): the analysis of technology of 1134-1148 (June 1999).

In a preferred embodiment, the analysis of kapillary heteroduplex can be used for detecting the existence of sudden change, this based on since the mobility of the double-strandednucleic acid in the capillary system that the existence of mispairing causes change.

From sample, produce the amplification that the nucleic acid that is used to analyze needs nucleic acid usually.Many amplification methods rely on enzymatic chain reaction (for example polymerase chain reaction, ligase chain reaction or the sequence replicating (self-sustained sequence replication) kept certainly) or depend on whole or a part of the duplicating of the carrier of being entered by the clone.Preferably, amplification of the present invention is the index amplification, and is shown as for example polymerase chain reaction.

The amplification method of many target spots and signal is narrated in the literature, for example, and Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic EngineeringNews 10:1, the generality that has provided these methods among the 54-55 (1990) is summarized.These amplification methods can be used to comprise polymerase chain reaction (PCR) in our method of invention, original position PCR (PCR in situ), ligase enzyme amplified reaction (LAR), ligase enzyme hybridization, Qbeta phage replication enzyme, based on the amplification system of transcribing (Transcription-based AmplificationSystem) (TAS), genome amplification is transcribed synchronous sequencing (genomic amplificationwith transcript sequencing) (GAWTS), based on the amplification technique (nucleic acid sequence based amplification) of nucleotide sequence (NASBA) and in situ hybridization.Be applicable to that the primer in the different amplification techniques can prepare according to known method in this technical field.

Polymerase chain reaction (PCR)

PCR is a nucleic acid amplification method, in U.S. Patent No. 4,683, is described among 195 and 4,683,202 grades.PCR is made of the recirculation of the primer extension reaction that archaeal dna polymerase produces.Target DNA is by thermally denature, and two oligonucleotide that target sequence is clipped in the middle on the relative chain of DNA to be amplified are hybridized.These oligonucleotide become primer, use jointly with archaeal dna polymerase.DNA obtains second double-stranded copy by primer extension and duplicates.By repeating the circulation of thermally denature, primer hybridization and extension, target DNA can be amplified 1,000,000 times or more in about two to four hours.PCR is a kind of biology tool, and it must determine amplification with the detection technique coupling.The superiority of PCR is that thereby it passes through in about 4 hours quantity with target DNA and increase 1,000,000 to 1,000,000,000 times and improve sensitivity.The diagnosis environment in, PCR can be used for increasing any known nucleic acid (Mok et al. (1994), Gynaecologic Oncology, 52:247-252).

From the sequence replicating of keeping (3SR)

From the sequence replicating of keeping (3SR) is the variant of TAS, it relates to by successive is several takes turns the isothermal duplication that ThermoScript II (RT), polysaccharase and nuclease carry out the nucleic acid masterplate, and these are active to mediate (Guatelli etal. (1990) Proc.Natl.Acad.Sci.USA 87:1874) by enzyme cocktail (cocktail) and suitable Oligonucleolide primers.Use the enzymatic degradation of the RNA in the RNA/DNA heteroduplex to replace heat denatured.RNase H and other all enzyme are added in the reaction, and all steps occur under the identical temperature, do not add other reagent.Follow this process, 10 ⁶To 10 ⁹Amplification just finish in 1 hour at 42 ℃.

Ligation amplification (LAR/LAS)

Ligation amplification reaction or ligation amplification system use dna ligase and four kinds of oligonucleotide, two kinds of oligonucleotide of every target chain.This method is at Wu, and D.Y. and Wallace describe among R.B. (1989) the Genomics 4:560.Oligonucleotide hybridization and links to each other by ligase enzyme to the flanking sequence of target DNA.This reaction is heated sex change, and circulation is repeated.

Q β replicative enzyme

In this technology, the rna replicon enzyme that duplicates the phage Q β of single stranded RNA is used to the target DNA that increases, as Lizardi et al., described in (1988) Bio/Technology 6:1197.At first, target DNA is hybridised on the primer that comprises T7 promotor and Q β 5 ' sequence area.In this process, use this primer, ThermoScript II produces and connects the cDNA that this primer arrives its 5 ' end.This two step is similar to the TAS testing program.The heteroduplex that generates is heated sex change.Next, use second primer that comprises Q β 3 ' sequence area to begin second and take turns the synthetic of cDNA.This step has produced the double-stranded DNA that comprises Q phagus beta 5 ' and 3 ' terminal and active T7 RNA polymerase binding site.The T7 RNA polymerase is transcribed the RNA that this double-stranded DNA is new simulation Q β then.Through fully the washing remove any not hybridization probe after, the RNA that elution makes new advances from this target duplicates by Q β replicative enzyme.Being reflected in about 20 minutes of back produced 10 ⁷Amplification doubly.

Alternative amplification technique can be used for the present invention.For example, rolling circle amplification (rolling circleamplification) (people such as Lizardi, (1998) Nat Genet 19:225) is a kind of commercial available amplification technique (RCAT ^TM), drive by archaeal dna polymerase, can under isothermal condition, adopt linearity or geometrodynamics duplicate the annular oligonucleotide probe.

Under two situations that the primer of design exists suitably, in 1 hour, produce 10 of each ring by DNA strand displacement and high branch (hyperbranching) ¹²Or more parts of copies, thereby realize the geometry amplification.

If use single primer, RCAT just produces the thousands of linear chain that connects the DNA copy of target successively in several minutes, and it is connected this target with covalent linkage.

Technology in addition, strand displacement amplification (strand displacement amplification) (SDA; People such as Walker, (1992) PNAS (USA) 80:392) from the exclusive specifically defined sequence of specific target.But the technology that is different from other dependence thermal cycling, SDA is a kind of isothermal process of using a series of primers, archaeal dna polymerase and restriction enzyme to come the unique nucleotide sequence of index amplification.

SDA comprises target generation phase and index amplification stage.

When producing target, the heating double-stranded DNA makes its sex change produce two strand copies.The primer of a series of special manufacturings and archaeal dna polymerase (the snubber primer (bumper primers) that is used to duplicate the amplimer of base sequence and is used to replace newly-generated chain) combine and produce the target of change that can the index amplification.

The index amplification procedure is a starting point with the target (the part single stranded DNA chain with restriction enzyme enzyme recognition site) of the change that the target generation phase produces.

Amplimer is incorporated on its complementary dna sequence on each bar chain.Then archaeal dna polymerase use primer discern from its 3 ' end extend the site of primer, use the target of change to be used to increase each Nucleotide as template.Therefore the primer that extends forms double chain DNA fragment, comprises the complete restriction enzyme enzyme recognition site of each end.

Restriction enzyme combines at its recognition site with double chain DNA fragment then.After cutting away a bilateral segmental only chain formation breach (nick), restriction enzyme breaks away from from recognition site.Archaeal dna polymerase discerns breach and from this site extended chain, displacement is the chain of formation in the past.Therefore this recognition site is repeatedly formed breach and recovery by restriction enzyme and archaeal dna polymerase, contains the segmental DNA chain of target and is replaced continuously.

Each metathetical chain can be used for annealing with amplimer then, as mentioned above.This process continues to carry out the displacement that multiple forms breach, extension and new DNA chain, and the result forms the index amplification of original DNA target.

In case nucleic acid is amplified, just can use many technology to detect the sudden change of single base pair.Wherein a kind of this type of technology be single strand conformation polymorphism (single stranded conformationalpolymorphism) (SSCP).SCCP detects based on the unusual migration of strand mutant DNA in the electrophoresis with respect to reference dna.Sudden change produces conformational change in single stranded DNA, cause mobility to change.Fluorescence SCCP uses fluorescently-labeled primer to be used for auxiliary detection.Therefore, use fluorescently-labeled primer increase reference and mutant DNA.Be amplified DNA by sex change and quenching to produce single strand dna, it detects by native gel electrophoresis.

Chemical mismatch cleavage (chemical mismatch cleavage) is (CMC) based on identification and the cracking of the combination of passing through azanol, perosmic anhydride and piperidines to the dna mismatch base pair.Therefore, reference dna and mutant DNA are by fluorescently-labeled primer amplification.Amplicon is hybridized, and uses the T base bonded perosmic anhydride with mispairing then, and perhaps the C base bonded azanol with mispairing carries out cracking, is that piperidines carries out cracking in adorned base site then.The cracked fragment is detected by electrophoresis then.

Can also use based on pvuii restriction fragment (restriction fragmentpolymorphism) technology (RFLPs).Although many single nucleotide polymorphism (singlenucleotide polymorphisms) (SNPs) do not allow to use traditional rflp analysis, primer inductive restriction analysis PCR (PIRA-PCR) to can be used for using the PCR primer to introduce restriction site in the mode that SNP-relies on.The primer that is used for PIRA-PCR of introducing suitable restriction site can design by computational analysis, and for example as Xiaiyi et al., (2001) Bioinformatics 17:838-839 is described.

In addition, the technology of analyzing based on WAVE can be used (Methods Mol.Med.2004; 108:173-88).This dna fragmentation analysis system can be used for detecting single nucleotide polymorphism, and it is based on the liquid chromatography and high resolution matrix (the Genet Test.1997-98 of temperature adjusting; 1 (3): 201-6)

PCR in real time (also claiming quantitative PCR, real-time quantitative PCR or RTQ-PCR) is method (the Expert Rev.Mol.Diagn.2005 (2:209-19) that the DNA of a kind of while quantizes and increases.By polymerase chain reaction, DNA is specifically increased.After each took turns amplification, DNA was quantized.Quantized common methods comprises fluorescigenic adorned DNA oligonucleotide (being called as probe) when using those fluorescence dyes that insert double-stranded DNA and those and complementary DNA to hybridize.

Be called as "

Primer " specific primer can be used to highly sensitive and DNA cloning system fast.Such primer combines probe in individual molecule with specific target sequence, produce to have the dynamic (dynamical) fluorescence detecting system of unit molecule (Nucl.Acids Res.2000; 28:3752-3761).The fluorescent probe system that this system is better than other is molecular lampmark (Molecular Beacons) and Taq for example

Part do not need to be the independent probe that combines with amplified target, make detect either rapider but also more efficient.Direct comparison (the Nucl.Acids Res 2000 of three kinds of detection methods; 28:3752-3761) point out

More excellent than the operation of intermolecular probe system, particularly under cycling condition fast.A certain version

The structure of primer is such, it is constrained in hairpin loop (hairpin loop) conformation by the complementary stem sequence (stem sequences) of about six bases, complementary stem sequence side be concerned about the specific probe sequence (Nat.Biotechnol.1999 of target; 17:804-807).Stem also is used for fluorescent reporter dye (being attached to 5 ' end) and quenching medium molecule are positioned in the very approaching zone.In this conformation, do not produce signal.The PCR-blocker is separated hairpin loop and primer sequence, forms

3 ' end.This blocker prevents to read over, and under the situation that does not have specific target, reads over and will cause separating of hairpin loop folding.During PCR, extend usually from primer.After sex change and annealing steps subsequently, hairpin loop is separated folding, and if correct product be amplified, in new synthetic chain, probe sequence combines in the primer downstream with specific target sequence.This new texture is more stable than original hairpin loop on thermodynamics.Fluorescent signal is produced now, because fluorescence dye no longer is in close proximity to quenching medium.Fluorescent signal is directly proportional with the quantity of target DNA.

Another

Primer comprises the complementary few nucleic acid duplex of two marks.An oligonucleotide in the two strands is by 5 ' terminal reporting dyes mark, and carries non-coding nucleotide of blocker and PCR primer element, and another oligonucleotide is by 3 ' end quenching agent dye marker.The principle of effect is then in essence with above-described

The hair clip primer is identical: during real-time quantitative PCR, 5 ' terminal reporting dyes and 3 ' end quenching agent dyestuff are separated from one another, cause the remarkable increase of fluorescent emission.

Can with amplification refractory mutation system (Amplification Refractory MutationSystem, ARMS) (Nucl.Acids Res.1989; 17:2503-2516, Nat.Biotechnol.1999; 17:804-807) unite and make single base mutation to be detected.Under suitable substance P CR condition, the single base mismatch that is positioned at primer 3 ' end is enough to satisfy the allelic preferential amplification of coupling fully people such as (, 1989) Newton, allows the differentiation between very relevant species.The basis of using the amplification system of above-described primer is that those oligonucleotide that have 3 ' residue of mispairing can not play the primer effect in PCR under suitable situation.This amplification system allows only by after agarose gel electrophoresis gene type being carried out in the inspection of reaction mixture.It is simple and reliable, and can clearly arbitrary allelotrope be come in the heterozygote and the homozygote difference of a certain locus.ARMS does not need digestion with restriction enzyme, the allele specific oligonucleotide of using usually or the sequential analysis of PCR product.

The collection of embodiment 1-clinical trial and serum sample

Carry out this research, the correlative study of testing as the clinical II of the multicenter phase of Gefitinib single therapy.The carrying out of this research, the recommendation based on the biomedical research Helsinki statement that relates to the human experimenter has obtained the approval of suitable ethics examination board (ethical review boards).The histology in IIIB or IV stage or cytology turn out to be chemotherapy- The Japanese patient of NSCLC participates in this test.Whole patient's orally give Gefitinib, fixed dosage is 250 milligrams of every days.Use guilding principle assessment validity (the J.Natl.Cancer Inst.2000 of " the response judgement criteria of solid tumor (Response Evaluation Criteria in SolidTumours, RECIST) "; 92:205-216).

28 patients were registered (table 1) on October 23rd, 2002 between 3 days Augusts in 2003.All patient's response is evaluated, and its existence that gets nowhere is tracked with overall existence.Gathered before beginning to give Gefitinib from 27 patients'blood samples (2 milliliters).The serum DNA concentration of extracting in all 27 duplicate samples is up to 1720ng/ml.

Sample collecting and DNA extraction.Gathered before beginning to give Gefitinib from 26 NSCLC patients'blood samples.Isolating serum is deposited until use in-80 ℃.Use Qiamp Blood test kit (Qiagen, Hilden, Germany) to extract and purified blood serum DNA, carry out according to the testing program of following modification.A pillar is used repeatedly, and is processed up to whole samples.The DNA that obtains goes out by the elution of the aseptic two distillation damping fluid of 50 μ l.The concentration of the DNA that extracts and purity are determined by spectrophotometry.The DNA that extracts is stored in-20 ℃ until use.

Embodiment 2-utilizes Scorpion primer and amplification refractory mutation system (ARMS) to examine Survey E746 A750 del and L858R EGFR sudden change

EGFR Scorpion ^TMThe susceptibility of test kit

Assess the susceptibility (Fig. 1) of EGFR Scorpion test kit by tentative experiment.When reaching maximum 45 circulation times, amount is from 1pg to 10, and (Fig. 1 a) in whole curves growths of the E746_A750del standard DNA of 000pg.When wild-type standard DNA and water were used as negative control, curve did not increase, and kept before reaching 50 cycle numbers that smooth (Fig. 1 a).Use the E746_A750del standard DNA and the wild-type standard DNA of dilution, ratio is 10 ⁰To 10 ^-5, when reaching maximum 45 cycle numbers, show to exist whole curves of E746_A750del to increase (Fig. 1 b).Typical curve in the measuring vol scope in this research is linear, r ²Value is 0.997 and 0.987.Two rate of curve almost parallel (Fig. 1 c).The Ct of E746_A750del standard DNA and the wild-type DNA of dilution only is that the Ct of E746_A750del standard DNA compares with those, and is all approaching in the E746_A750del of each amount standard DNA.Although in ratio less than 10 ^-3The time, the E746_A750del standard DNA of dilution and the peak fluorescence level of wild-type DNA be not than there being the low of wild-type DNA standard, but the existence of E746_A750del can clearly be detected.Reach the influence that the curve of the E746_A750del DNA of 1pg amount is not sneaked into by Wild type EGFR DNA at most.In the test of using human cancer cell line's genomic dna, as expected, use the signal of the DNA that derives from the PC-9 cell to be detected, from not being detected of A431 cell based on cell.

We use and combine ARMS ^TMAnd Scorpion ^TMThe EGFRScorpion of these two kinds of technology ^TMTest kit (DxS company limited, Manchester, Britain) detects the sudden change in the real-time PCR reactions.Being used for detecting four kinds of scorpion primers in E746_A750del, the L858R of exon 19 and exon 21 and wild-type is designed by DxS Ltd. (Manchester, Britain) and synthesizes.Be used for the human EGFR sequence (registration number: AY588246) that the sequence of the scorpion primer of E746_A750 del and L858R is filed based on GenBank-.All are reflected in the 25 μ l volumes carries out, and comprises that 1 μ l template DNA, 7.5 μ l react buffer mixture, 0.6ml primer mixed solution and 0.1ml Taq polysaccharase.Whole reagent is included in this test kit.PCR in real time is used Smart

II (Cepheid, Sunnyvale CA) carries out, according to following condition: carry out initial sex change 10 minutes at 95 ℃, the fluorescence reading at 95 ℃ 30 seconds, 62 ℃ of 50 round-robin 60 seconds and each circulation end (be arranged on FAM, allow optical excitation to measure) at 480nm and at 520nm.Use Cepheid SmartCycler software (Ver.1.2b) to carry out data analysis.Threshold circulation (threshold cycle) (Ct) is defined in second circulation of peak-peak of deriving curve, and it has represented the maximum curvature point of growth curve.Ct and maximum fluorescence (Fl) are used for result's analysis.Positive findings is defined as: Ct≤45 and Fl 〉=50.These analyses in each sample are carried out two parts.In order to confirm to detect the sensitivity of E746_A750del, we use the standard DNA that comprises in the EGFR Scorpion test kit.Usage quantity is 1,10,100,1,000 or 10, the standard DNA of 000pg with E746_A750del, and 10, the wild-type standard DNA of 000pg and amount are respectively the mixture of the E746_A750del standard DNA of 1,10,100,1,000 or 10,000 pg.In order to quantize,, obtain typical curve with of the log value mapping of Ct cycle number to the DNA amount of known standard.Linearly dependent coefficient (R ²) numerical value and the formula of slope calculated.Be used for Japanese gland cancer PC-9 clone and the known human epithelium cancer A431 clone that comprises exons 19 wild-types of the DNA extraction of positive control from the known E746_A750del of comprising, 10,000pgDNA is used.

Detect the EGFR mutation status of serum DNA by ARMS

The E746_A750del or the L858R that pick up from 27 patients' NSCLC serum DNA are examined.The wild-type of exons 19 and exon 21 all is detected in whole serum samples.E746_A750del is detected in 12 patients' sample.L858R is detected (table 2) in a patient.Fully, the EGFR sudden change is detected in 27 patients' 13 (48.1%).The histology hypotype of original tumour of carrying the patient of EGFR sudden change in 23 serum is summarized among the table 3a.Sudden change is positive for EGFR in 11 (47.8%) in 23 gland cancer cases, 2 the squamous cell carcinoma cases 1 and 2 the large cell carcinoma cases 1.EGFR mutation status and histological type are irrelevant on the statistics.The EGFR sudden change that derives from female patients is than derive from more being detected of the male sex (in 10 7,70% in the sample of being everlasting; Corresponding to 6 in 17,29.4%, table 3b).

EGFR mutation status and for the response of Gefitinib in the serum

From display part response (PR) or stable disease (SD) (in 17 cases 11,75%) the EGFR sudden change in the patient samples is recently from PD (PD, in 10 cases 2,18%) is observed (p=0.046 significantly more continually in the patient samples, Fisher ' s exact test, table 3c).

Embodiment 3: in the serum EGFR mutation status and for existence influence

Statistical analysis.Fisher ' s exact test is used for relatively having different qualities, comprises sex, tumor type and for the response of Gefitinib, patient NSCLC in the existence of EGFR sudden change.About the analysis for the response of Gefitinib, according to the RECIST standard, patient is divided into partial response or stable disease (PR/SD) and two groups of PD (PD).We use standard log-rank test, the Kaplan-Meier curve of more totally surviving and getting nowhere and surviving." overall existence " (OS) is defined as from beginning to give the time of Gefitinib to the death that is caused by any reason; The known still patient of survival is examined in their last follow-up study when analyzing." existence (PFS) gets nowhere " is defined as from beginning to give the time of Gefitinib to the death that PD occurs for the first time or caused by any reason; Known survival and do not have the patient of PD in their last follow-up study, to be examined when analyzing.0.05 the P value be considered to significant statistically.Use the Stat View routine package of version 5.0 to carry out statistical study.

The PFS intermediate value of accepting whole patients of Gefitinib treatment is 98 days, and the OS intermediate value is 306 days.The patient who carries EGFR sudden change in the serum compares with the patient who does not have the EGFR sudden change, have significantly longer PFS intermediate value (in contrast to 46 days in 200 days, P=0.005, Fig. 3 is a).The patient who carries EGFR sudden change compares with the patient who does not have the EGFR sudden change, has longer OS intermediate value, although there is not statistical significance (611 days with respect to 232 days, P=0.078, Fig. 3 b).These results suggest Serum EGF concentration R sudden change is played the effect of prognostic factor for the existence that gets nowhere with totally surviving, simultaneously can also be as the predictor (predictor) of patient for the response of Gefitinib treatment.

Embodiment 4: by direct order-checking and with ARMS relatively come EGFR in the serum analysis Sudden change

Direct order-checking (direct sequence) by the serum DNA of extraction in 10 (37.0%) from 27 patients detects deletion sudden change (E746_A750del).

Pcr amplification and directly order-checking.Each sample that obtains from serum and tissue samples is carried out two parts of amplifications and directly order-checking.In 25 μ l volumes, carry out PCR, use the Ampli Taq Gold archaeal dna polymerase (Perkin-Elmer of 15 μ l template DNAs, 0.75 unit, RocheMolecular Systems Inc., Branchburg, NJ), 2.5 μ l PCR damping fluids, every kind of primer of 0.8mMdNTP, 0.5 μ M and the MgCl that depends on the different concns of polymorphism sign ₂The sequence of primer set and the program of amplification are followed as previously described (Nuc.AcidsRes.1989; 17:2503-2516) carry out.(Perkin-Elmer, FosterCity CA) increase to use thermal cycler.(Applied Biosystems, Foster City CA) checks order to use ABI prism 310.Human sequence's (registration number: AY588246) compare that sequence and GenBank file at EGFR.

Point mutation in the exons 18,19,20 and 21 does not detect in the PCR product from serum sample.Serum EGF concentration R state that detects by direct order-checking and histological type, sex, all unconnected statistically for the response (table 3) and the survival advantage (PFS: P=0.277, OS: P=0.859, supplementary data 2) of Gefitinib.15 (55.6%) in the EGFR mutation status that obtains by direct order-checking and 27 paired sampleses (paired samples) by ARMS obtain consistent.EGFR sudden change (E746_A750del) in 4 cases is positive by direct order-checking, is negative by ARMS.8 cases are negative by direct order-checking, are positive by ARMS.

Embodiment 5: the comparison of EGFR sudden change in EGFR sudden change and the serum in the tumour

Look back, 20 tumor samples obtain from 15 patients.

Tissue sample is gathered and DNA extraction.The acquisition of tumor specimen is according to carrying out through the testing program of Ethics Committee (Institutional Review Board) approval.Look back, before treatment, be collected from 20 paraffin masses of 15 patient's tumor materials and be used for diagnosis.By TBLB (trans bronchiallung biopsy), the tumor sample of 11 primary carcinoma is gathered, 1 by excision, and 9 come from transitivity site (4 come from bone, 3 come from lymphoglandula, 1 come from brain and 1 and come from colon).All sample is through the diagnosis of histological examination with confirmation NSCLC.Use DEXPAT ^TMTest kit (TaKaRa Biomedicals, Shiga, Japan) carries out the DNA extraction of tumor sample.

EGFR exons 19 carries out under identical PCR condition with 21 order-checking.From 12 patient's tumor samples by check order (table 4).In 4 cases, detect EGFR sudden change (25.0%); In 3 cases wherein in the exon 19 deleted 15bp (E746_A750del), and exon 21 is L858R in 1 case.Histological type with patient of EGFR sudden change: 3 is that gland cancer and 1 are large cell carcinomas.These 4 patients are for the response of Gefitinib: 2 patients are PR, and 1 patient is SD, and 1 patient is PD.Other 3 samples do not have evaluated owing to the amplification of PCR product is too low.

Look back, several tumor sample and serum sample are picked up from 11 patients (table 4).In paired sample, consistent in the EGFR mutation status of 8/11 (72.7%) tumour and the serum.E746_A750del in two patient's tumours be positive and serum in be negative, the E746_A750del in patient's tumour be negative and serum in be positive.

Table 1 patient characteristics

PS, behavior state; Ad, gland cancer; Scc, squamous cell carcinoma; Large, large cell carcinoma; PR, partial response; SD, stable disease; PD, PD.

Table 2 patient characteristics and the EGFR mutation status that uses EGFR ARMS-Scorpion method from serum DNA, to detect

SD, stable disease; PD, PD; PR, partial response; M, the male sex; F, the women; Ad, gland cancer; Large, large cell carcinoma; Scc, squamous cell carcinoma; +, the detected curve of SmartCycler;-, not the detected curve of SmartCycler;

Table 3 is according to histology (a), sex (b) with for the response (c) of Gefitinib, the frequency of the EGFR sudden change among the lung cancer patient serum DNA.28 patients of overall 27 sample sources before treatment.

A histology and EGFR mutation status

B sex and EGFR mutation status

C is for the response and the EGFR mutation status of Gefitinib

Ad, gland cancer

PR, partial response; SD, stable disease; PD, PD;

EGFR mutation status in table 4 tumor sample and the serum sample.Several tumour and serum sample are derived from 12 patients.

M, the male sex; F, the women; SD, stable disease; PD, PD; PR, partial response; Scc, squamous cell carcinoma; Ad gland cancer; Large, large cell carcinoma

* the patient who has the different states EGFR sudden change of the DNA that originates from the DNA and the serum in tumour source.

Table 5

Protein

Position wild-type sudden change

688 L P

694 P L/S

709 E K

709 E V

715 I S

720 S F

718 L P

719 G S/C/A/D

724 G S

730 L F

733 P L

735 G S

742 V A

delE746_A750

delE746_S752V

delE746_P753insLS

delL747_A750insP

delL747_T751insP

746 E K

del750_754

751 T I

752 S Y

755 A P

del756_758

761 D N

768 S I

769 V L

770 D N

772 H L

772 P S

773 V M

776 R C

778 G F

781 C R

783 T I

784 S F

790 T M

792 L P

798 L F

810 G S

820 Q STOP

826 N S

834 V M

835 H Y

836 R C

847 T I

850 H N

851 V A

853 I T

857 G R

858 L M

858 L R

859 A T

861 L Q

863 G D

864 A T/V

866 E K

873 G E

877 P S

880 W STOP

882 A T

893 H Q

895 S G

897 V I

958 R P

Nucleotide

2063 C T

2080 C T

2081 C T

2118 C T

2125 G A

2126 A T

2142 G A

2144 T G

21?53 T C

2155 G T/A/C

2156 G C/A

2159 C T

2169 C T

2170 G A

2188 C T

2198 C T

2203 G A

2225 T C

2236 G A

del2247_2262

2252 C T

del2268_2275

2281 G A

2303 T G

2305 G C

2308 G A

2314 C T

2326 C T

2340 C T

2341 T C

2348 C T

2351 C T

2364 C T

2369 C T

2375 T C

2392 C T

2406 C T

2428 G A

2421 C T

2458 C T

2477 A G

2484 G A

2500 G A

2502 G A

2503 C T

2506 C T

2508 C T

2523 G A

2540 C T

2548 C A

2552 T C

2553 C T

2563 A T

2565 G A

2569 G A

2570 G T

2571 G T

2572 C A

2575 G A

2582 T A

2588 G A

2588 T C

2590 G A

2591 C T

2596 G A

2607 C T

2618 G A

2629 C T

2639 G A

2644 G A

2676 C T

2679 C A

2683 A G

2689 G A

2877 A G

Sequence table

SEQ?ID?NO.1

<SEQ01；DNA；HOMO?SAPIENS>3633

ATGCGACCCTCCGGGACGGCCGGGGCAGCGCTCCTGGCGCTGCTGGCTGCGCTCTGCCCGGCGAGTCGGGC

TCTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCACGCAGTTGGGCACTTTTGAAGATCATT

TTCTCAGCCTCCAGAGGATGTTCAATAACTGTGAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAG

AGGAATTATGATCTTTCCTTCTTAAAGACCATCCAGGAGGTGGCTGGTTATGTCCTCATTGCCCTCAACAC

AGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCCT

TAGCAGTCTTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGAAATTTACAGGAA

ATCCTGCATGGCGCCGTGCGGTTCAGCAACAACCCTGCCCTGTGCAACGTGGAGAGCATCCAGTGGCGGGA

CATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATGGACTTCCAGAACCACCTGGGCAGCTGCCAAAAGT

GTGATCCAAGCTGTCCCAATGGGAGCTGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATC

ATCTGTGCCCAGCAGTGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGC

TGCAGGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCTGCCGCAAATTCCGAGACGAAGCCACGTGCA

AGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGATGTGAACCCCGAGGGCAAA

TACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGCGT

CCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGC

CTTGCCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAAT

ATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGA

CTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAG

GGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATA

CGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATT

ACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATA

CAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAG

GGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG

AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAAC

ATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGT

CAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATG

TGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAAT

GGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGG

GATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGAGAGGG

AGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACT

GAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGTTCGGCACGGTGTATAAGGGACTCTGGATCCCAGA

AGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGG

AAATCCTCGATGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGC

CTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACA

CAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAGGGCATGAACTACTTGG

AGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAG

ATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGT

GCCTATCAAGTGGATGGCATTGGAATCAATTTTACACAGAATCTATACCCACCAGAGTGATGTCTGGAGCT

ACGGGGTGACTGTTTGGGAGTTGATGACCTTTGGATCCAAGCCATATGACGGAATCCCTGCCAGCGAGATC

TCCTCCATCCTGGAGAAAGGAGAACGCCTCCCTCAGCCACCCATATGTACCATCGATGTCTACATGATCAT

GGTCAAGTGCTGGATGATAGACGCAGATAGTCGCCCAAAGTTCCGTGAGTTGATCATCGAATTCTCCAAAA

TGGCCCGAGACCCCCAGCGCTACCTTGTCATTCAGGGGGATGAAAGAATGCATTTGCCAAGTCCTACAGAC

TCCAACTTCTACCGTGCCCTGATGGATGAAGAAGACATGGACGACGTGGTGGATGCCGACGAGTACCTCAT

CCCACAGCAGGGCTTCTTCAGCAGCCCCTCCACGTCACGGACTCCCCTCCTGAGCTCTCTGAGTGCAACCA

GCAACAATTCCACCGTGGCTTGCATTGATAGAAATGGGCTGCAAAGCTGTCCCATCAAGGAAGACAGCTTC

TTGCAGCGATACAGCTCAGACCCCACAGGCGCCTTGACTGAGGACAGCATAGACGACACCTTCCTCCCAGT

GCCTGAATACATAAACCAGTCCGTTCCCAAAAGGCCCGCTGGCTCTGTGCAGAATCCTGTCTATCACAATC

AGCCTCTGAACCCCGCGCCCAGCAGAGACCCACACTACCAGGACCCCCACAGCACTGCAGTGGGCAACCCC

GAGTATCTCAACACTGTCCAGCCCACCTGTGTCAACAGCACATTCGACAGCCCTGCCCACTGGGCCCAGAA

AGGCAGCCACCAAATTAGCCTGGACAACCCTGACTACCAGCAGGACTTCTTTCCCAAGGAAGCCAAGCCAA

ATGGCATCTTTAAGGGCTCCACAGCTGAAAATGCAGAATACCTAAGGGTCGCGCCACAAAGCAGTGAATTT

ATTGGAGCATGA

<SEQ02；PROTEIN/1；HOMO?SAPIENS>1210

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQ

RNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQE

ILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKI

ICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGK

YSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATN

IKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEII

RGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN

SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMN

ITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTN

GPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKET

EFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGIC

LTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVK

ITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEI

SSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTD

SNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSF

LQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNP

EYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEF

IGA

Claims (33)

1. be used for detecting the method for the one or more sudden changes of ErbB acceptor :-

(a) provide biologicfluid sample from patient;

(b) from described sample, extract DNA; With

(c) screen described DNA at the existence of one or more sudden changes in this receptor.

2. the method for claim 1 comprises and detects one or more sudden changes in the ErbB acceptor, and this sudden change changes the activity of Tyrosylprotein kinase in the described acceptor.

3. method as claimed in claim 1 or 2, wherein this ErbB acceptor is EGFR.

As before the described method of each claim, be used to predict the response of patient for the ErbB receptor agents, comprise step :-

(a) provide biologicfluid sample from patient;

(b) from described sample, extract DNA; With

(c) screen described DNA at the situation that exists that changes one or more sudden changes of tyrosine kinase activity in this receptor.

5. as each described method in the claim 1 to 3, be used to monitor the response of patient, comprise step for the ErbB receptor agents :-

(a) provide biologicfluid sample from patient;

(b) from described sample, extract DNA; With

(c) screen described DNA at the situation that exists that changes one or more sudden changes of tyrosine kinase activity in this receptor.

6. method as claimed in claim 4, wherein cancer patient has been foretold this patient's survival advantage for the prediction of the response of ErbB receptor agents.

7. method as claimed in claim 4 further comprises step:

(d) conclude: be detected the patient that carries mutant and wild-type allele for the response of ErbB receptor agents with positive, and the patient who only is detected wild-type allele can not make the male response to this medicine.

As before the described method of each claim, wherein screening method comprises the use polymerase chain reaction, this polymerase chain reaction is used to detect single base mutation, little in-frame deletion or the allele-specific primers of base replacement.

9. method as claimed in claim 8, wherein screening method relates to and uses real-time polymerase chain reaction (real time-PCR), and this real-time polymerase chain reaction uses and detects the allele-specific primers that single base mutation, little in-frame deletion or base are replaced.

10. method as claimed in claim 8 or 9, wherein first primer is to being used to detect wild-type allele, and second primer is to being used to detect mutation allele; Wherein every centering primer comprises :-

(a) has the primer that sports allele specific 3 ' terminal nucleotide to specific; With

(b) the possible additional mispairing of this primer 3 ' end.

11. method as claimed in claim 10, wherein every centering primer comprises :-

(a) contain primer sequence and to the unit molecule or the nucleic acid duplex probe of other special sequences of target sequence;

(b) the fluorescence report dyestuff that be attached to probe 5 ' end close within described unit molecule or nucleic acid duplex with the quenching medium molecule;

(c) at the one or more non-coding nucleotide residue of described probe one end;

(d) wherein, described report dyestuff separates during the amplification of target sequence with the quenching medium molecule.

12. method as claimed in claim 11, wherein this probe is

Probe.

13. as before the described method of each claim, wherein by using the technology can detect the mutant nucleotide sequence that exists with wild-type sequence 10% level to detect sudden change.

14. as before the described method of each claim, wherein this biofluid is any in blood, serum, blood plasma, sweat or the saliva.

15. method as claimed in claim 11, wherein this biofluid is a serum.

16. as before the described method of each claim, wherein this ErbB receptor agents is the ErbB receptor tyrosine kinase inhibitors.

17. as before the described method of each claim, wherein this ErbB receptor agents is the EGFR tyrosine kinase inhibitor.

18. method as claimed in claim 16, its Chinese traditional medicine is selected from by Gefitinib, erlotinib (Te Luokai, OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinibditosylate (GW2016, GW-572016, GSK572016), canertinib (CI-1033, PD183805), AEE788, XL647, BMS 5599626, ZD6474 (Zactima ^TM) or WO2004/006846 or WO2003/082290 in the group that constitutes of disclosed any compound.

19. method as claimed in claim 15, wherein most preferred EGFR tyrosine kinase inhibitor is Gefitinib or Tarceva.

20. method as claimed in claim 14, wherein the ErbB receptor agents is an anti-egfr antibodies, be selected from by Cetuximab (Erbitux, C225), the group that constitutes of Herceptin (EMD-72000), handkerchief Buddhist nun monoclonal antibody (ABX-EGF/rHuMAb-EGFR), MR1-1, IMC-11F8 or EGFRL11.

21. as before the described method of each claim, wherein the ErbB receptor agents be used as single therapy or with the use of uniting of other medicines.

22. as before the described method of each claim, wherein the sudden change be insertion, disappearance or the replacement of nucleic acid.

23. method as claimed in claim 22, wherein sudden change betides the tyrosine kinase domain of ErbB acceptor.

24. as before the described method of each claim, wherein sudden change betides the tyrosine kinase domain of EGFR.

25. method as claimed in claim 22, wherein sudden change bunch combines near the ATP-binding site of EGFR exons 18,19,20 or 21.

26. method as claimed in claim 22, wherein sudden change is selected from the group of the EGFR sudden change that is listed in the table 5.

27. method as claimed in claim 24, wherein sudden change is E746_A750del in the EGFR exons 19 and the L858R in the EGFR exon 21.

28. as before the described method of each claim, wherein the cancer that patient suffered from is selected from by non-noumenal tumour for example leukemia, multiple myeloma or lymphoma, and the noumenal tumour group that constitutes of bile duct, bone, bladder, brain/CNS, glioblastoma, breast, colorectum, uterine cervix, uterine endometrium, stomach, head and neck, liver, lung, muscle, neurone, oesophagus, ovary, pancreas, pleura/peritonaeum, prostate gland, kidney, skin, testis, Tiroidina, uterus and external genital tumor for example.

29. method as claimed in claim 4 further comprises step:

(d) screen described DNA at the situation that exists of the one or more sudden changes in the element of ErbB acceptor downstream signal pathway.

30. composition, it comprises first primer of being used to detect ErbB acceptor wild-type allele to right with second primer that is used to detect mutation allele, and wherein, a primer of every centering further comprises:

(a) has the primer that specific sudden change is had allele specific 3 ' terminal nucleotide;

(b) the possible additional mispairing of this primer 3 ' end;

(c) comprise primer sequence and to the unit molecule or the nucleic acid duplex probe of the special other sequence of target sequence;

(d) the be attached to 5 ' terminal fluorescence close with the quenching medium molecule within described unit molecule or nucleic acid duplex is reported dyestuff;

(e) at the one or more non-coding nucleotide residue of described probe one end;

(f) wherein, described report dyestuff separates during the target sequence amplification with the quenching medium molecule.

Be used for predicting the purposes in analysis that biofluid carry out of patient 31. be specific to the primer of ErbB acceptor for the response of ErbB medicine.

32. be specific to the purposes of primer in making composition of ErbB acceptor, thereby said composition is used for the response of test organisms fluid prediction patient for the ErbB medicine.

33. purposes as claimed in claim 31 further comprises step:

(a) from described sample, extract DNA; With

(b) screen described DNA at the situation that exists that changes one or more sudden changes of tyrosine kinase activity in the ErbB acceptor.

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